Hyperforin induces Ca(2+)-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A(2) activation through select phospholipid interactions.
ABSTRACT Here, we investigated the modulation of cytosolic phospholipase A(2) (cPLA(2))-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10microM) versus unstimulated cells, which was blocked by cPLA(2)alpha-inhibition, and induced translocation of cPLA(2) to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca(2+) by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA(2) at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA(2) phosphorylation. However, neither AA release nor translocation of cPLA(2) was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA(2) in the presence of Ca(2+). However, when Ca(2+) was omitted, hyperforin caused a prominent increase in cPLA(2) activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional (1)H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA(2) activation by enabling its access towards select lipid membranes independent of Ca(2+) ions. Such Ca(2+)- and phosphorylation-independent mechanism of cPLA(2) activation may apply also to other membrane-interfering molecules.
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ABSTRACT: Hyperforin is a plant derived antibiotic from St. John's wort. Here we describe a novel activity of hyperforin, namely its ability to inhibit the growth of tumour cells by induction of apoptosis. Hyperforin inhibited the growth of various human and rat tumour cell lines in vivo, with IC(50) values between 3-15 microM. Treatment of tumour cells with hyperforin resulted in a dose-dependent generation of apoptotic oligonucleosomes, typical DNA-laddering and apoptosis-specific morphological changes. In MT-450 mammary carcinoma cells hyperforin increased the activity of caspase-9 and caspase-3, and hyperforin-mediated apoptosis was blocked by the broad-range caspase inhibitor zVAD.fmk. When added to MT-450 cells, hyperforin, but not paclitaxel, induced a rapid loss of the mitochondrial transmembrane potential Deltapsi(m), and subsequent morphological changes such as homogenization and vacuolization of mitochondria. Monitoring of Deltapsi(m) revealed that the hyperforin-mediated mitochondrial permeability transition can not be prevented by zVAD.fmk. This indicates that mitochondrial permeabilization is a cause rather than a consequence of caspase activation. Moreover, hyperforin was capable of releasing cytochrome c from isolated mitochondria. These findings suggest that hyperforin activates a mitochondria-mediated apoptosis pathway. In vivo, hyperforin inhibited the growth of autologous MT-450 breast carcinoma in immunocompetent Wistar rats to a similar extent as the cytotoxic drug paclitaxel, without any signs of acute toxicity. Owing to the combination of significant antitumour activity, low toxicity in vivo and natural abundance of the compound, hyperforin holds the promise of being an interesting novel antineoplastic agent that deserves further laboratory and in vivo exploration.Oncogene 03/2002; 21(8):1242-50. · 7.36 Impact Factor
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ABSTRACT: Cholesterol-rich membrane domains, which contain the scaffold protein caveolin-1 (Cav-1) (caveolae), represent an important structural element involved in endothelial signal transduction. The present study was designed to investigate the role of these signaling platforms in the generation of endothelial-derived hyperpolarizing factor (EDHF). Caveolae were disrupted by cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD 10 mM). MbetaCD-induced modulation of non-nitric oxide-/non-prostanoid-dependent (EDHF)-mediated vasorelaxation was studied in pig coronary arteries. Effects of MbetaCD on endothelial Ca(2+) signaling and phospholipase A(2) (cPLA(2)) activity were determined using fura-2 imaging and measurement of [(3)H]-arachidonate mobilization in cultured pig aortic endothelial cells (PAEC). Cellular localization of caveolin-1 and phospholipase A(2) was investigated by cell fractionation, and interaction of cPLA(2) with caveolin-1 was tested by immunoprecipitation experiments. MbetaCD inhibited EDHF-mediated relaxations of pig coronary arteries induced by bradykinin (100 nM) or ionomycin (300 nM) but not relaxations induced by the NO donor DEA/NO (1 microM). Exposure of arteries to cholesterol-saturated MbetaCD failed to affect EDHF-mediated relaxations. Cholesterol depletion with MbetaCD did not affect bradykinin or ionomycin-induced Ca(2+) signaling in pig aortic endothelial cells, but was associated with enhanced basal and reduced Ca(2+)-dependent release of arachidonic acid (AA). Cell fractionation experiments indicated targeting of cPLA(2) to low density, caveolin-1 rich membranes and immunoprecipitation experiments demonstrated association of phospholipase A(2) with the scaffold protein of caveolae, caveolin-1. Cholesterol depletion with MbetaCD did not disrupt the interaction between cPLA(2) and caveolin-1 but prevented targeting of cPLA(2) to low density membranes. Exogenous supplementation of arachidonic acid after cholesterol depletion partially restored EHDF responses in pig coronary arteries. The integrity of caveolin-1-containing membrane microdomains is prerequisite for arachidonic acid recruitment and EDHF signaling in porcine arteries.Cardiovascular Research 12/2004; 64(2):234-42. · 5.94 Impact Factor
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ABSTRACT: The action of various purified phospholipases on intact cells is reviewed, with special emphasis on platelets. Their effects are strongly dependent on the substrate specificity of the enzymes. Moreover, phospholipases can be divided into two groups according to their ability to hydrolyse phospholipids in an intact cell. Comparative studies of their behaviour towards phospholipid monolayers maintained at different surface pressures have allowed to estimate the surface pressure of erythrocyte and platelet membrane as 31–34 dynes/cm.Agents and Actions 11/1979; 9(4):400-6.